Project Summary/Abstract: Advanced thyroid cancer patients continue to have dismal prognosis and poor survival ratees due to lack of effective therapies. While the MAPK pathway accounts for a majority of the mutations that arise in thyroid cancer (BRAF, RAS, RET/PTC), targeting this pathway has had mixed success in the clinic. The recent approval of a BRAF inhibitor in combination with a MEK1/2 inhibitor for anaplastic thyroid cancer patients (ATC) with a BRAF V600E mutation is monumental, however, this combination is only effective in subset of ATC patients, and is ineffective in BRAF-mutant papillary thyroid cancer (PTC) patients; highlighting the necessity to identify new therapeutic strategies for patients who do not respond to current therapies. Our lab has discovered Src as a clinically relevant target in thyroid cancer and we have demonstrated that Src inhibition with two structurally independent Src inhibitors, dasatinib and saracatinib, inhibits growth, invasion, and metastasis. However, like many single-agent targeted therapies, clinical responses to single-agent Src inhibition has been limited in solid tumors, indicating combination therapies will be necessary. Accordingly, recent studies have shown that the Src inhibitor, dasatinib, in combination with chemotherapeutic agents has clinical benefit, thus supporting the rationale to identify targets that can be co-inhibited with Src. To this end, our lab has demonstrated that co-inhibition of Src and MEK1/2 synergizes to inhibit growth, increase survival, and induce apoptosis in BRAF- and RAS-mutant thyroid cancer cells, while PIK3CA-mutant cells were shown to be resistant to this combination. To begin to decipher mechanism(s) mediating sensitivity in response to combined Src and MEK1/2 inhibition, I used a reverse phase protein array (RPPA) of >400 proteins and phosphoproteins and identified the pro-apoptotic protein, BIM, as a potential mediator of response. Therefore, the goals of my proposal are to determine the regulation of apoptosis by cooperative Src and MAPK signaling in thyroid cancer. Specifically, in Aim 1 I will determine the regulation and role of PI3K/AKT-dependent signaling in mediating the apoptotic response, in Aim 2 I will determine the mechanism(s) of apoptosis regulated by Src and MAPK signaling, and finally in Aim 3, I will determine the role of combined Src and MEK1/2 inhibition on tumor growth, metastasis, and the induction of apoptosis in vivo. This proposal emphasizes a molecular understanding of mechanisms mediating response to Src and MEK1/2 inhibition to develop new strategies to enhance these effects and potential approaches to sensitize resistant tumors. The completion of the proposed research will help the National Cancer Institute fulfill their mission to support cancer research and training in the fundamental sciences.